CD45 is a leukocyte-specific transmembrane glycoprotein whose intracellular domain exhibits protein tyrosine phosphatase (PTP) activity. CD45 is critically involved in several signal transduction pathways including antigen receptor-mediated activation of T and B lymphocytes. However, the mechanism of CD45-mediated signal transduction remains unclear. Its physiological substrates and the factors influencing its PTP activity need to be clarified. We have recently shown that phosphorylated protein of 30 kDa (CD45-AP) is specifically and strongly associated with CD45. By virtue of its association with CD45, we have been able to purify CD45-AP, microsequence it, and obtain its mouse cDNA. There is not significant homology with already known sequences. Expression of CD45-AP appears to be restricted to cells which express CD45 (i.e. leukocytes). We hypothesize that CD45- AP acts as an "adapter" which directs the interaction between CD45 and other molecules involved in the CD45-mediated signal transduction pathway. In order to test this hypothesis, we propose to utilize the CD45-AP cDNA clone in characterizing the role of CD45-AP in the various aspects of CD45-mediated lymphocyte signal transduction. Specific Aims: (1) Prepare antibodies to CD45-AP (2) Determine the cell types which express CD45-AP (3) Characterize the interaction of CD45 with CD45-AP by determining a. Subcellular localization of CD45 and CD45-AP b. Molecular associations of CD45-AP c. Phosphorylation state of CD45 and CD45-AP d. Correlation between the PTP activity of CD45 and its association with CD45-AP in resting lymphocytes and lymphocytes stimulated by the CD45-mediated signal transduction pathway (4) Block CD45-AP synthesis by antisense oligonucleotides and determine the effect on various cellular events triggered by the CD45-mediated signal transduction pathway (5) Overexpress CD45-AP by transfecting lymphocytes with a CD45-AP expression vector and determine the effect on various cellular events triggered by the CD45-mediated signal transduction pathway Long-term goal: To understand the mechanism of signal transduction in CD45-expressing cells and roles of CD45 in leukocyte differentiation and immune responses.